The regulation of gene expression by p53 in response to various cellular stresses is extremely complex, as is the regulation of important p53-dependent downstream functions: cell cycle arrest, apoptosis and DNA repair. Despite the huge body of information regarding the accumulation, activation and function of p53, much remains to be discovered. This proposal has two distinct aims. Specific Aim 1: Characterize the MDM2-independent control of p53 stability and transcriptional activity. We have found that UV radiation and some other stimuli induce the accumulation of p53 in the complete absence of the major negative regulator MDM2. We will use a variety of methods to delineate this novel regulatory mechanism. Furthermore, in the absence of MDM2, camptothecin does not change p53 levels at all but nevertheless does activate p53 to drive gene expression. This useful way to uncouple changes in p53 levels from changes in the intrinsic activity of the protein will be exploited to learn more about the gene-specific effects of specific p53 phosphorylations. Specific Aim 2: Identify novel regulators of p53 by using forward genetics. Our strategy involves abrogation of the p53- dependent constitutive expression of the thymidine kinase and puromycin resistance genes, driven by the constitutive upregulation of p53 in response to activated N-ras in HT 1080 cells. The basic cell line has already been used successfully to isolate five different mutant cell lines. Two new strategies involve both chemical and insertional mutagenesis, the latter to mark genes whose disruption leads to the selected phenotype. The identification of novel regulators of p53 and its target proteins has great potential to contribute to our understanding of the amazing complexity of p53 activation and function. [unreadable] [unreadable]